Throughout this application, various publications, including United States patents, are referenced by author and year and patents by number. The disclosures of these publications and patents and patent applications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
Traumatic brain injury (TBI) is a major cause of death and severe disability, with an estimated incidence of 1.5 million new cases per year in the United States, unfortunately resulting in 50,000 fatalities. A total of 5.3 million Americans, approximately 2% of the U.S. population, currently live with disabilities resulting from TBI. Activation of coagulation within the central nervous system (CNS) is an early and nearly universal event accompanying severe TBI. The brain is rich in tissue factor, the initiator of the coagulation cascade [Goodnight, S. H. et al. N. Engl. J. Med. 290:1043-1047 (1974)], which is released in response to TBI. Clot formation in the systemic circulation is evident within minutes and likely occurs even earlier at the site of injury [Stein, S. C. et al. J. Neurosurg. 97:1373-1377 (2002)]. There is a strong correlation between the extent to which coagulation is activated, the development of progressive changes seen on CT scan, and the likelihood of an adverse outcome, that is not dependent on the severity of the injury alone [Stein, S. C. et al. Neurosurgery 32:25-30; discussion 30-21(1993)]. Indeed, severe coagulopathy is second only to shock as an independent predictor of adverse outcome.
Plasminogen activators are enzymes that activate the zymogen plasminogen to generate the serine proteinase plasmin, which degrades fibrin. Among the plasminogen activators studied are streptokinase, urokinase and human tissue plasminogen activator (tPA) and dismutase.
Tissue type plasminogen activator (tPA) a multidomain, glycosylated, serine protease is a fibrin specific activator of plasminogen and a very effective thrombolytic agent. tPA is a protein whose primary application is in the treatment of heart attack and stroke patients. First characterized in 1979, as an important and potent biological pharmaceutical agent in the treatment of various vascular diseases due to its high fibrin specificity and potent ability to dissolve blood clots in vivo. Beechem Laboratories clot dissolving product-anisoylated plasminogenstreptokinase activator complex, trade named, Eminase, is claimed to reduce the death rate in heart attack victims by 50%. Gene Tech's TPA (Activase), is also highly effective in dissolving blood clots.
Natural tPA has a plasma half-life of about six minutes or less. Due to its rapid clearance from the circulation, tPA has to be infused to achieve thrombolysis. Front loaded dosing with increased concentrations of tPA has shown more rapid and complete lysis compared to the standard infusion protocol and early potency is correlated with improved survival rate.
Plasminogen Activators and TBI
The continued existence of fibrin clots is regulated not only by exposure of tissue factor, but also by the fibrinolytic system. Fibrin is lysed by plasmin. Plasmin is formed from the proenzyme plasminogen through cleavage of a single peptide bond, primarily by tissue type (tPA) or urokinase type (uPA) plasminogen activator. The activity of plasmin is restrained by the formation of enzymatically inactive complexes with the circulating inhibitor α2-antiplasmin, while tPA and uPA are regulated in a similar manner by forming complexes with plasminogen activator inhibitor-1 (PAI-1). These complexes are rapidly cleared from the circulation by the LDL receptor-related protein (LRP).
Transgenic mice deficient in endogenous tPA (tPA−/−) have the propensity to accumulate fibrin spontaneously [Carmeliet, P. et al. Nature, 369:419-424 (1994)] and are less able to lyse clots added exogenously [Carmeliet, P. et al. (1994) Ibid.; Bdeir, K. et al. Blood, 96:1820-1826 (2000)]. Yet, tPA−/− mice exhibit smaller cortical lesions and less edema after TBI and have smaller lesions and better recovery of neurological function after spinal injury [Abe, Y. et al. Journal of Neurotrauma, 20:43-57 (2003)] than WT mice. In models of stroke initiated by mechanical occlusion, the size of the infarcted area in tPA−/− mice is also smaller than in WT animals [Wang, Y. et al. Nat Med, 4:228-231 (1998)], whereas in models characterized by cerebral thrombosis tPA deficiency exacerbates cerebrovascular fibrin deposition and CNS injury [Tabrizi, P. et al. Arterioscler. Thromb. Vasc. Biol., 19:2801-2806 (1999)]. These observations suggest that tPA may have deleterious effects on CNS function in the setting of TBI, notwithstanding the benefit attributable to its fibrinolytic activity.
Cerebral Vasoactivity of tPA and its Possible Neurotoxic Effects
The present inventors have previously reported that IV injection of tPA decreases cerebral vascular resistance in rats, and therefore tPA alters vascular tone. The inventors have previously found that high concentrations (>20 nM) of tPA stimulate the contraction of isolated aortic rings and increase systemic blood pressure in rats [Nassar, T. et al. Blood, 103:897-902 (2004)]. In contrast, tPA at these and all concentrations tested induced vasorelaxation in the cerebral circulation of pigs and rats. The ensuing decrease in cerebral vascular resistance (CVR) may be linked to the development of cerebral edema, one of the more serious complications of stroke and revascularization of ischemic areas induced by tPA. In support of this notion is the finding that tPA−/− mice develop less brain edema after TBI.
NMDA Receptor in TBI and Effect of tPA on Cerebral Hemodynamics
The N-methyl-D-aspartate receptor (NMDA) is an ionotropic receptor that binds the excitatory amino acid transmitter glutamine. Activation of NMDA-R elicits cerebral vasodilation and may represent a mechanism that couples local metabolism to blood flow [Faraci, F. B., K R. Circulation Research. 1993, 72:476-480 (1993)]. All glutamate receptor subtypes have been implicated in neurotoxicity. However, the NMDA subtype is thought to play a crucial role in excitotoxic neuronal cell death [Choi, D. Journal of Neurobiology, 23:1261-1276 (1992)]. Glutamatergic system hyperactivity has been demonstrated in animal models of TBI, and NMDA-R antagonists protect against experimental brain injury [Katayama Y, B. D. et al. Journal of Neurosurgery, 73:889-900 (1990); Kawamata, T. K. et al. Journal of Cerebral Blood Flow and Metabolism, 12:12-24 (1992)]. tPA is reported to signal within the CNS by cleaving the NR-1 subunit of NMDA-R [Nicole O, D. F. et al. Nat. Med., 7:59-64 (2001)].
In earlier studies, the inventors examined the role played by the glutamate NMDA receptor (NMDA-R) in tPA-mediated CNS injury post-TBI. The results of these experiments showed that the PAI-1 derived peptide EEIIMD (also denoted by SEQ ID NO. 3) inhibits the NMDA-R mediated vasoactivity including that post TBI.
Taking into consideration that tPA exerts its effect on the NMDA-R by cleaving its NR-1 subunit, the inventors speculated that mutant tPA that lacks the catalytic activity, may compete with the endogenous tPA that is released and reaches high concentrations in the CSF during TBI. This competitive effect may reduce or even prevent tPA induced neurotoxicity.
Moreover, tPA is known as having a limited therapeutic-window, when used for therapy. Specifically, tPA improves the clinical outcome in patients, but only if administered within three hours of the onset of an ischemic stroke in human patients. In addition to its brief therapeutic window, the need for radiological definition of stroke size and etiology and the increased incidence of symptomatic intracerebral hemorrhage (ICH) has constrained its clinical use. Furthermore, as also indicated above, several studies suggest that tPA may increase (directly or indirectly) post-stroke neuronal degeneration. In contrast, the mutated tPA molecule of the invention demonstrates no limited therapeutic window. As shown by FIG. 2, administration of the mutated tPA molecule of the invention to CHI animal model even two hours (corresponds to ten hours in human), resulted in significant beneficial neuroprotective effect as reflected by improved NSS, specifically as compared to saline controls and to the deleterious effect of the WT tPA on the NSS outcome.
Alterations in glutamate production may further contribute to secondary head injury. Glutamate levels, increased clinically after TBI [Choi, D. (1992) Ibid.; Katayama Y, B. D. et al. (1990) Ibid.], may affect other paths related to secondary head injury including the initiation of apoptosis by activation of NMDA-R, calcium dependent production of nitric oxide and development of superoxides and free radical damage to DNA and cellular membranes. Intact blood brain barrier (BBB) after TBI or stroke may contribute to accumulation of glutamate and is assumed to have a deleterious effect on brain function. tPA increases the permeability of the BBB and thus facilitates the clearance of neurotoxic agents such as glutamate. This effect of tPA on BBB is mediated through the LRP (LDL receptor-related protein is LRP) and has been shown by others as requiring its catalytic activity.
Surprisingly, and in contrast to Yepes et al. [Yepes et al. J. Clin. Invest. 112:1533-1540 (2003)], the inventors have now found that the mutated tPA molecule of the invention lacking any catalytic activity is still capable of mimicking some of the extracatlytic activities of WT tPA, particularly that of increasing the permeability of the BBB. Thus, clearly showing for the first time that the effect of tPA on reducing the integrity of the BBB, is not related to its catalytic activity. This mutant has been shown by the inventors as promoting the clearance of the neurotoxic amino acid glutamate and of lactate from the CSF, and thereby impeding their accumulation post TBI and also augmenting the salutary effect of the mutant on neurological outcome. These results raises the possibility that transient disruption of the BBB in the area of injury by the mutant tPA contributes directly to the improved outcome of TBI. That stands in opposition to widely accepted views in the field.
The object of the present invention therefore, is to provide a mutated tPA molecule devoid of serine protease catalytic activity, having ability to increase permeability of the BBB, and thereby facilitating the clearance of neurotoxic amino acids and of lactate, as a safe molecule having a wide therapeutic-window, for use in the treatment, amelioration and prevention of stroke and conditions involving neuronal damage, more particularly, acute brain injury and neurodegenerative disorders.
These and other objects of the invention will become clearer as the description proceeds.